The present invention relates to a method of producing transgenic tomatoes having an increased free amino acid content, and transgenic tomatoes thus produced. In particular, the present invention relates to a method of producing transgenic tomatoes containing at least one of asparagine, aspartic acid, serine, threonine, alanine, histidine and glutamic acid accumulated in a large amount, and transgenic tomatoes produced by this method.
The technique of transforming a plant by introducing a particular gene thereinto was reported for the first time in the world in the study wherein it was achieved by introducing a gene into tobacco with Agrohacterium tumefaciens, a soil bacteria. Thereafter, many products having useful agricultural characteristics were produced, and it was also tried to produce useful components in plants. A plant breeding method by such a recombinant DNA technique is considered to be hopeful in place of the ordinary, traditional technique of developing varieties of plants. In this field, improvement in the characteristics of plants concerning nitrogen assimilation is also being studied. The study of amino acids is particularly prospering because they are important ingredients of particularly fruits, root crops and seeds and also they exert a great influence on the tastes of them.
Reports on the biosynthesis of amino acids include, for example, a report that free lysine content of tobacco was increased to 200 times as high content by introduction of DHDPS gene derived from E. coli into tobacco (U.S. Pat. No. 5,258,300, Molecular Genetics Res. and Development); a report that free lysine content was increased by introduction of AK gene (EP 485970, WO 9319190); a report that asparagine content was increased to 100 times as high content by introduction of AS gene into tobacco; and a report that tryptophan content was increased to 90 times as high content by introduction of an anthranilic acid-synthesizing enzyme into a rice plant (WO 9726366, DEKALB Genetic Corp). The plants into which a gene is to be introduced are not limited to model plants such as tobacco and Arabidopsis thaliana but plants which produce fruits such as tomato are also used. For example, as for tomatoes, a transformant thereof was obtained by Agrobacterium co-cultivation method in 1986 [S. McCormick, J. Niedermeyer, J. Fry, A. Barnason, R. Horsch and R. Freley, Plant Cell Reports, 5, 81-84 (1986); Y. S. Chyi, R. A. Jorgenson, D. Goldstern, S. D. Tarksley and F. Loaiza-Figueroe, Mol. Gen. Genet., 204, 64-69 (1986)]. Since then, investigations were made for the improvement of the recombinant DNA techniques. Various genes relating to the biosynthesis of amino acids and nitrogen assimilation other than those described above are also known. They include asparaginase and GOGAT, and the nucleotide sequences of them were also reported.
Glutamic acid which is one of xcex1-amino acids is widely distributed in proteins. It is generally known that a tasty component of tomatoes used as a seasoning and also a tasty component of fermentation products of soybeans (such as soy sauce and fermented soy paste) are glutamic acid. It is also known that glutamic acid is synthesized in the first step of nitrogen metabolism in higher plants. It is also known that glutamine and asparagine formed from glutamic acid are distributed to tissues through phloem and used for the synthesis of other amino acids and proteins. It was reported that in plants, glutamic acid exists in a high concentration in phloem through which photosynthesis products such as sucrose and amino acids are transported [Mitsuo Chino et al., xe2x80x9cShokubutsu Eiyo/ Hiryogakuxe2x80x9d p. 125 (1993)]. As for examples of glutamic acid contained in a high concentration in edible parts of plants, it is known that about 0.25 g/100 gf. w. of glutamic acid is contained in tomato fruits [xe2x80x9cTokimekixe2x80x9d No. 2, Nippon Shokuhin Kogyo Gakkaishi, Vol. 39, pp. 64-67 (1992)]. However, glutamic acid of a high concentration cannot be easily accumulated in plant bodies because it is a starting material for amino group-donors and also it is metabolized in various biosynthetic pathways as described above even though the biosynthesizing capacity of the source organs can be improved.
As far as the applicant knows, it was not yet succeeded to remarkably increase glutamic acid concentration in edible parts of plants by either cross breeding or gene manipulation. For example, although transgenic plants were produced by the introduction of GDH (glutamate dehydrogenase) gene, it was reported that when glutamate dehydrogenase GDH (NADP-GDH) gene derived from Escherichia coli was introduced into tobacco and corn for the purpose of imparting resistance to phosphinothricin used as a herbicide, glutamic acid content of the roots of them was only increased to 1.3 to 1.4 times as high [Lightfoot David et al. CA2180786 (1966)]. Namely, in this report, glutamic acid content of tobacco roots was merely increased from 14.7 mg/100 gf. w. to 20.6 mg/100 gf. w., and that of corn roots was increased from 16.2 mg/100 gf. w. to 19.1 mg/100 gf. w. Although there are other reports on the use of GDH gene, no example is given therein [WO 9509911, xcex1,xcex2-subunits derived from chlorella (WO 9712983)]. In addition, no analytical value of amino acids of glutamic acid group was given therein.
In the first step of the assimilation of inorganic nitrogen into an organic substance, ammonia is incorporated into glutamic acid for mainly forming glutamine. This process is catalyzed by glutamine synthetase enzyme (GS). Then glutamine is reacted with xcex1-ketoglutaric acid in the presence of glutamate synthase (GOGAT) to form two molecules of glutamic acid. This GS/GOGA cycle is considered to be the main pathway of the nitrogen anabolism in plants [Miflin and Lea, Phytochemistry 15; 873-885 (1976)]. It is known that the ammonia anabolism proceeds also by a metabolic pathway other than the pathway wherein ammonia incorporation is catalyzed by GS [Knight and Langston-Unkefer, Science, 241: 951-954 (1988)]. Namely, in this metabolic pathway, ammonia is incorporated into xcex1-ketoglutaric acid to form glutamic acid. This process is catalyzed by glutamate dehydrogenase (GDH). However, plant GDH has a high Km value for ammonia. The role of this pathway under normal growing conditions has not yet been elucidated enough because ammonia is toxic and the concentration of intracellular ammonia is usually low. A researcher reported that this pathway contributes to the nitrogen anabolism when intracellular ammonium concentration is increased over a normal level (Knight and Langston-Unkefer, supra).
Glutamic acid thus synthesized is further utilized for the synthesis of other amino acids such as asparagine, alanine, phenylalanine, leucine, isoleucine, glycine, valine, serine, tyrosine, proline and xcex3-aminobutyric acid (GABA). It is particularly known that GABA is accumulated in storage organs such as tomato fruits and sugar beat roots. From this fact, the consumption of glutamic acid is supposed. It is also known that the accumulation of GABA is induced by environmental stresses such as the acidity in the cell, low temperature and heat shock [Streeter and Thimpson, Plant Physiol., 49, 572-578 (1972): Raggiani et al., Plant Cell Physiol., 29; 981-987 (1988): Menengus et al., Plant Physiol., 90; 29-32 (1989): Roberts et al., Plant Physiol., 98; 480-487 (1992): Shelp et al., Plant Physiol., 94; 219-228 (1995): Aurisano et al., Plant Cell Physiol. 36; 1525-1529 (1995): Wallace et al., Plant Physiol., 75; 170-175 (1990): and Mayer et al., Plant Physiol., 94; 796-810]. GABA is synthesized from glutamic acid by the catalyzing function of glutamate decarboxylase (GAD). The activity of GAD is controlled by intracellular Ca(calcium) ion concentration and calmodulin [Ling et al., Plant Cell, 6; 1135-1143 (1994): Snedden et al., Plant Physiol., 108; 543-549 (1995): Arazi et al., Plant Physiol., 108; 551-561 (1995): and Snedden et al., J. Biol. Chem, 271; 4148-4153 (1996)]. It is considered that the change in the intracellular Ca ion concentration is induced by various stresses to rapidly increase GAD activity. As a result, the action of GABA as a signal-transducing substance in plant cells is expected, but the details thereof have not yet been elucidated.
A gene encoding GAD was isolated from a petunia for the first time [Baum et al., J. Biol. Chem., 268; 19610-19617 (1995)]. Then GAD gene was isolated from tomatoes [Gallego et al., Plant Mol. Biol., 27; 1143-1151 (1995)] and Arabidopsis [Zik et al., Plant. Mol. Biol., 37; 967-975 (1998)]. In all the cases, calmodulin-binding site comprising 30 to 32 amino acids is present at C-terminal in common. However, the organs where the gene expression was found vary depending on kinds of the plants. In petunias, the organs are flower petals and flowers and in tomatoes, the organs are fruits. It was also reported that GAD 1 genes were expressed in roots and GAD 2 genes were expressed in the whole plant body of Arabidopsis.
Gideons et al [EMBO, 15; 2988-2996 (1996)] introduced GAD genes isolated from petunia into tobacco to obtain a transformant for the purpose of investigating regulatory function of calmodulin on GAD expression. According to their report, they made two kinds of tests. In one of the tests, the whole GAD gene length was introduced in the sense direction and in another test, the introduction was conducted in the same manner but the calmodulin-binding site was removed. The transformants were examined to find that GABA was increased and glutamic acid was decreased in amount in each cases. The degree of the change in amount was higher in the case in which calmodulin-bonding site was removed. It was also reported that the height of the plants was shortened and morphologic difference was observed.
The object of the present invention is to provide a method of increasing free amino acid contents of storage organs of plants, in particular, a method of increasing at least one of free amino acids selected from the group consisting of glutamic acid, asparagine, aspartic acid, serine, threonine, alanine and histidine contained in edible parts such as roots and seeds of plants; and also to provide transgenic plants in which said free amino acids are accumulated in a large amount.
The object of the present invention is attained by providing a plant having a changed expression level of a major enzyme concerning the assimilation and utilization of nitrogen and/or a plant having an organ-specific expression, and a method of producing such a plant. The plant can be produced by introducing at least one gene encoding an enzyme which assimilates or utilizes nitrogen together with a suitable regulatory sequence and excessively expressing the same or controlling the expression thereof.
In particular, by introducing a glutamate decarboxylase (GAD) gene in antisense direction into the plant, free amino acids, particularly at least one of glutamic acid, asparagine, aspartic acid, serine, threonine, alanine and histidine are accumulated in a large amount in the plant.
Therefore, in a preferred embodiment, the method of the present invention comprises the step of
a) trasnforming a plant with a genetic construct containing an antisense sequence of a gene sequence encoding glutamate decarboxylase (GAD), a regulatory sequence capable of expressing the antisense sequence and a marker gene,
b) selecting or identifying the transgenic plant base on a character imparted by the marker gene in the genetic construct;
c) screening the transgenic plant in which one or more free amino acids are accumulated in a larger amount; and
d) selecting the transgenic plant in which one or more free amino acids are accumulated in a larger amount.
The present invention relates to the manipulation of a gene concerning nitrogen metabolism in plants. In particular, the present invention relates to a method of changing the expression level of an enzyme responsible for the assimilation and utilization of nitrogen for the purpose of accumulating a free amino acid, particularly glutamic acid which improves taste, in a large amount in edible parts such as fruits, roots (of root crops) and seeds of useful plants. By increasing, modifying or controlling the expression level of such an enzyme, plants having desired properties can be obtained.